Oncolytic virotherapy with the adenovirus mutant dl 1520 in combination with chemotherapy has shown clinical response. Approaches to cancer gene therapy involving delivery of enzymes to activate the prodrugs CB 1954 and 5-FC are currently being tested in clinical trials. We hypothesised that the combination of an adenoviral vector equivalent to dl1520 with activation ofCB 1954or 5-FC and the combination of CB 1954 activation with 5-FU may further improve the antitumour effects for colorectal cancer therapy. The initial in vitro data showed that the combination of dl 1520 with CB 1954 activation or 5-FU (metabolite of 5-FC activation) and the combination of CB 1954 activation with 5-FU led to an additive or synergistic cytotoxicity. Subsequent data showed that the incorporation of Ntr or CD-UPRT genes into replicating oncolytic adenoviruses (ROAds) resulted in enhanced Ntr expression or CD-UPRT activity and augmented cytotoxic effects in tissue culture, surpassing the levels and cytotoxic effects mediated by the corresponding replication-defective vectors. When tested in subcutaneous human colon cancer xenografis, Ntr expression mediated by the ROAd was apparently higher than the level mediated by replication-defective CTLI02. Importantly, the antitumoural efficacy of CB 1954 activation mediated by ROAd is significantly superior to that mediated by CTLI 02 (p = 0.01). The ROAds displayed viral replication and oncolysis in vitro and in vivo and these attributes can contribute to the increased gene expression level and enhanced efficacy. Overall, the data suggested that the use of ROAds improved Ntr or CD-UPRT expression and antitumoural efficacy in the presence of corresponding prodrugs and may have the potential to achieve clinical significance in the treatment for colorectal cancer.

Conditionally-replicating adenoviruses (CRAds) have generally demonstrated only modest therapeutic efficacy in human clinical trials, in part due to their poor ability to spread throughout a tumor mass. In these studies, I first examined whether inclusion of an intact early region 3 (E3) and the p14 fusion-associated small transmembrane (FAST) protein in a CRAd vector can enhance oncolytic efficacy by improving viral spread. E3 encodes the adenovirus death protein (ADP), which enhances virus progeny release from infected cells, while p14 FAST can allow spread of the virus through cell-cell fusion. I generated viruses with (CRAdRC109) or without (CRAdRC111) an intact E3 region, which encoded the p14 FAST gene between the fiber coding sequence and E4 region of their viral genomes. In the A549 human lung cancer cell line, both CRAdRC109 and CRAdRC111 expressed p14 FAST at very low levels when compared to CRAdFAST, a similar virus that expressed the protein from within the E3 deletion, and thus had a relatively poor ability to mediate cell-cell fusion. Although inclusion of E3/ADP in CRAdRC109 did result in larger plaques and increased virus spread relative to CRAdRC111, neither virus showed improved oncolytic activity relative to CRAdFAST. I subsequently developed CRAdRC116, in which the E3 region of the viral genome was replaced with a bicistronic expression cassette containing the p14 FAST and ADP coding sequences separated by a self-cleaving 2A peptide sequence. This virus co-expressed p14 FAST and ADP and caused extensive cell-cell fusion in A549 cells. However, expression of ADP from CRAdRC116 did not increase cancer cell killing nor virus spread, and thus did not enhance oncolytic efficacy relative to CRAdFAST. These studies suggest that p14 FAST and ADP do not exhibit synergy when co-expressed from a CRAd vector. Future studies should instead focus on combining other methods of improving viral spread in conjunction with expression of ADP or FAST proteins from CRAd.

Many oncolytic virotherapies have shown great advantages for rapid, rational design through recombinant DNA technology to facilitate the targeting of a broad spectrum of malignancies. Newcastle disease virus (NDV), an avian paramyxovirus, is naturally tumor-selective and inherently oncolytic. Our approach is to develop NDV-based nanoparticles (VBNP) for oncolytic virotherapy. VBNPs are non-infectious and non-replicating and are relatively safe. We obtained VBNPs by co-expressing matrix (M), hemagglutinin (HN), and fusion (F) proteins of NDV in avian/ mammalian cells. The budding characteristics, size and morphology of VBNPs were similar to authentic virions. As a proof of concept, we engineered the apoptin (VP3) gene of chicken anemia virus in VBNPs and specifically targeted them to folate-receptor bearing tumor cells by surface conjugation to folate. The VBNPs killed tumor cells by apoptosis and induced proinflammatory and chemotactic cytokines. The VBNPs, although not curative, were able to limit the progression of xenotransplanted fibrosarcoma and malignant glioma tumors and provided a survival advantage in nude mice. We also engineered NDV M based particles with nipah virus surface glycorporteins to target ephrin B receptors. NDV based nipah Virus BNPs (NiV-ndBNP) were morphologically similar to authentic NiV virions. NiV glycoproteins were incorporated into the NDV M based particles, despite poor sequence homology in the transmembrane domain and cytoplasmic tails of glycoproteins. Our results suggest that VBNPs could be used to deliver small molecules, tumor antigens, anti-tumor/ reporter genes and also aid in generating tumor specific immunity by rational design. / Master of Science

No description available.

Oncolytic virotherapy is emerging as a new treatment option for cancer patients. At present, there are relatively few oncolytic virus clinical trials that are underway or have been conducted, however one virus that shows promise in pre-clinical models is Vesicular Stomatitis Virus (VSV). VSV is a naturally occurring oncolytic rhabdovirus that has the ability to preferentially replicate in and kill malignant versus normal cells. VSV also has a low seroprevalence, minimal associated morbidity and mortality in humans, and simple non-integrating genome that can be genetically manipulated, making it an optimal oncolytic vector. Currently, many labs are using a variety of different strategies including inserting trans genes that can modulate the innate and adaptive immune response. VSV can also be retargeted by altering its surface glycoprotein (G) or be made replication incompetent by deleting the G protein. Currently, our lab has engineered a series of new recombinant VSVs, incorporating either the murine p53 (mp53), IPS-1, or TRIF transgene. mp53, IPS-1 and TRIF were incorporated into the normal VSV-XN2 genome and mp53 was also incorporated into the mutated VSV-ΔM vector generating VSV-mp53, VSV-IPS-1, VSV-TRIF and VSV-ΔM-mp53. Our data using these new viruses indicate that these viruses preferentially replicate in and kill transformed versus non-transformed cells and efficiently express the transgene. However, despite the ability for VSV-IPS-1 and VSV-TRIF to induce a robust type 1 IFN response, VSV-ΔM-mp53 was the only construct that had reduced toxicity and elicited an increased anti-tumor response against a syngeneic metastatic mammary tumor model. VSV- ΔM-mp53 treatment lead to a reduction in IL-6 and IP-10 production, an increase in tumor specific CD8+ T cells, and immunologic memory against the tumor. Collectively these studies highlight the necessity for additional VSV construct development and the generation of new clinically relevant treatment schema.

The central aim of this thesis was to develop strategies to improve the manufacture of the group B chimeric oncolytic adenovirus, ColoAd1, which rapidly kills and lyses host cells. In attempting to improve the cellular yield of ColoAd1, this thesis therefore sought to identify host infection-related factors that limited ColoAd1 production. In the widely-used manufacturing cell line, HEK293, ColoAd1 replication depleted intracellular ATP earlier than Ad11p and activated the intracellular energy sensor, AMPK. This might have reflected earlier ATP depletion, or possibly the absence of the E4orf4 protein from ColoAd1 compared to Ad11p. Despite this difference in AMPK activation, both viruses appeared able to maintain mTORC1 activity, which may be essential particularly for protein synthesis in the early stages of virus infection. For production purposes, preventing intracellular ATP depletion was seen as an attractive mechanism of maintaining ColoAd1 infected host cell viability and was hypothesised to lead to increased virus yield. A range of strategies were explored to enhance depleting ATP levels. Even though none of these were dramatically successful, they indicated that perhaps the anabolic building blocks required for viral replication were more important than cellular energy levels. Finally, a screening methodology based on siRNA knockdown was used to identify kinases that affected ColoAd1 replication. Many hits were identified, and several candidate kinases indicated a role for intracellular calcium signalling limiting virus particle production. Overall, data presented in this thesis supports the manufacture of ColoAd1 in HEK293 cells and suggest that enhancing glycolysis may increase ColoAd1 yield. It also provides mechanistic insights into the replication of ColoAd1 and Ad11p that may inform the improved design of group B oncolytic adenoviruses.

No description available.

The use of intraperitoneal (i.p.) adenovirus virotherapy of ovarian cancer is currently limited by insufficient efficacy and high toxicity. Both factors are associated with adenovirus serotype 5 (Ad5) in this setting and may be serotype-specific. Low levels of uptake receptors (CAR and αV integrins) on ovarian tumour cells and widespread immunity against Ad5 among patients appear to restrict efficacy and intraperitoneal inflammatory responses against Ad5 were among the reasons for the termination of a phase II/III clinical trial in ovarian cancer. This thesis sought to overcome these obstacles by investigating the alternative adenovirus serotypes Ad3 and Ad11. For these viruses lower pre-existing antiviral immunity and utilisation of different uptake receptors have been reported. Furthermore, virus cloaking with novel polymers which could impart enhanced protection from neutralisation was examined. In vitro, wild-type Ad3, Ad5 and Ad11 displayed differential oncolytic activity in a panel of ovarian cancer cell lines which partly correlated to uptake receptor expression and virus internalisation. However, some cell lines displayed lysis resistance in a serotype-specific manner. While the inflammatory response six hours after i.p. administration of Ad11 in CD46-transgenic mice did not differ from Ad5, in long-term studies of repeated administration Ad5 induced significantly more severe pathologic effects in the form of adhesions and liver toxicity than Ad11 or mock-treatment. Oncolysis inhibition assays using malignant exudate samples demonstrated greater neutralisation of Ad3 and Ad5 in comparison to Ad11 at low concentrations of samples. Notably, 10-fold less Ad11 than Ad5 was required for oncolytic efficacy at a sample concentration of 10%. In an ex vivo model of ascites from ovarian cancer patients Ad5 modified with novel polymer formulations achieved at least 50% cell kill in six of eight samples, in contrast to two of eight samples for non-modified Ad5. These data suggest that virotherapy using Ad11 might be advantageous over Ad3 or Ad5. The lack of strong inflammation and the possibility to decrease treatment doses due to less neutralisation of Ad11 might result in considerably improved patient safety. Chemical modification of Ad with novel polymers presents an exciting advancement in overcoming treatment neutralisation in adenovirus virotherapy.

MicroRNAs are small non-coding RNA molecules that regulate mRNA translation by binding to complementary sequences usually within the 3’ un-translated region (UTR). By inserting four perfectly complementary binding sites for the hepatic specific microRNA mir122 into the 3’ UTR of adenovirus wild type 5 (Ad5 WT) E1A mRNA I show that the acute liver toxicity caused by Ad5 WT in mice can be significantly reduced. This virus, termed Ad5-mir122, is a promising virotherapy candidate and causes no obvious liver pathology whilst maintaining Ad5 WT replication in mir122 negative cells. Data shows that repeat intravenous administration of Ad5-mir122 (2x1010vp) to HepG2 tumour bearing mice mediated significant anti-cancer efficacy. RT-QPCR for E1A mRNA demonstrated a 29-fold reduction when compared to Ad5 WT in murine liver whilst western blot confirmed that all E1A protein variants were knocked down. Viral genomic replication was also reduced in mouse liver by 25-fold compared to Ad5 WT. This control of virus activity reduced alanine and aspartate transaminase release by >15-fold and histological analysis showed little to no pathology in Ad5-mir122 infected livers. Measurement of mature mir122 levels in Ad5-mir122 infected livers by RT-QPCR showed that the quantity of mir122 remained unaffected at therapeutic doses. Complete genome mRNA array profiling of infected livers showed that the transcript levels of >3900 different mRNAs were changed more than 2-fold following Ad5 WT infection whilst less than 600 were changed by Ad5-mir122. A non-replicating control adenovirus vector altered >550 mRNAs. No known mir122 target mRNAs were affected following infection with Ad5-mir122. Western blot analysis of a known mir122 regulated target (Aldolase A) confirmed these results, demonstrating no change in protein level despite infection with Ad5-mir122. These data combined demonstrate that the exploitation of microRNA mir122 regulation to control adenovirus replication is a safe method of control and does not alter the endogenous level or activity of the microRNA or its endogenous mRNA targets. Ad5-mir122 is a potent anti-cancer agent that replicates to wild-type levels in microRNA mir122 negative cells but is specifically and safely attenuated in hepatocytes.

Les virus oncolytiques sont une nouvelle classe d’agents thérapeutiques pouvant être une alternative au traitement des cancers. Plusieurs virus oncolytiques sont actuellement développés en clinique, néanmoins de nombreuses améliorations sont à apporter afin de créer une nouvelle classe de virus plus efficaces et moins toxiques. Le premier objectif de cette thèse a été d’améliorer la spécificité tumorale du virus de la vaccine via le ciblage de l’antigène MUC1 présenté à la surface des cellules tumorales. Pour cela un virus recombinant présentant à sa surface un fragment d’anticorps (scFv) dirigé contre l’antigène tumoral MUC1 a été construit et produit. Les tests in vitro n’ont toutefois pas permis de mettre en évidence un ciblage spécifique du virus recombinant. Un deuxième aspect de cette thèse a été de tester le potentiel oncolytique de virus de la famille des Poxviridae. Durant ce travail de thèse, les capacités oncolytiques de douze poxvirus, appartenant à 8 genres différents, ont été étudiés. Leurs effets sur la prolifération de cellules cancéreuses humaines ont été évalués. Les virus caractérisés par un effet oncolytique élevé ont été, par la suite, modifiés et armés par ingénierie virale afin d’augmenter leur efficacité. La dernière partie de cette thèse a été consacrée à la génération et la sélection de virus chimériques basées sur la méthode d’évolution dirigée. Cette méthode est utilisée pour mimer le processus naturel de sélection évolutif. Appliqué à la virothérapie oncolytique, ce procédé nous a permis de générer un nouveau virus oncolytique chimérique caractérisé par un potentiel anti-cancéreux amélioré. En résumé, cette thèse a permis, par des techniques d’ingénierie virale, par un criblage de nouveaux virus et par la méthode d’évolution dirigée, de créer et de sélectionner une nouvelle génération de poxvirus oncolytiques présentant une activité thérapeutique accrue avec un profil de toxicité atténué et pouvant être utilisés dans diverses indications thérapeutiques. / Oncolytiques viruses are a new class of therpeutic agents which could be an alternative for cancer treatment. Currently, several oncolytic viruses are evaluated in clinical trial, nevertheless improvements are needed to create a new class of more efficiente and less toxic viruses. The first objective of this thesis was to improved the vaccinia virus specificity through the targeting of the tumor-associated antigen MUC1. To address this goal, a recombinant virus expressing an scFv targeting the MUC1-protein was engineered and produced. However, in vitro, the demonstration of a specific targeting by the recombinant virus was not possible. A second aspect of this thesis work was to evaluate the oncolytic potential of Poxviridae family viruses. Oncolytic capacities of twelve viruses, belonging to eight genera, were evaluated. Their impact on human cancer cells was tested. In order to increase their efficacity, viruses with the highest oncolytic capacities were then modified and armed by genetic engineering. The third part of this work was devoted to the generation of chimeric viruses based on directed evolution process. This methodology is used to mimic the natural process of evolutionary selection. Applied to oncolytic virotherapy, this technique allowed the generation of a new chimeric oncolytic virus caracterised by an enhanced antitumoral potential. In summary, this thesis has allowed, through viral engineering, poxviruses screening and directed evolution methodology, the creation and selection of a new generation of oncolytic poviruses. These viruses demonstrate an increased therpeutic activity and greatest safety profil enabling their application in several therapeutic indication.